Amino acid trace analysis method for impure protein in glucose polymer

Abstract

The invention discloses an amino acid trace analysis method for impure protein in a glucose polymer, which is characterized in that the content of amino acid after acidolysis of impure protein is used as a detection object, and the content of amino acid is measured by solid phase extraction-acidolysis-precolumn derivatization-high performance liquid chromatography. The method can effectively determine the content of 18 amino acids after acidolysis of the impure protein. The detection method disclosed by the invention is specific, reliable, universal, good in recovery rate, high in sensitivity and accuracy and wide in detection range, and plays a positive role in safety research and quality control of the medicinal raw material medicines or auxiliary materials.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, in particular to the detection of amino acid content, and specifically to a method for amino acid trace analysis of impurity proteins in glucose polymers. Background Art

[0002] Some high molecular weight glucose polymers, such as dextran 20, dextran 40 and dextran 70, are prepared by the fermentation process of the strain of Leuconostoc pseudomesenteroides. The finished products often contain residual protein substances introduced by the process. This kind of protein is called host cell protein (HCP) in the field of biopharmaceuticals. If it exceeds a certain amount, it may cause immune response or allergic reaction in patients. It is regarded as process impurity in Europe and the United States. Therefore, the detection of HCP residue is the most commonly performed analysis project in drug research and development and quality control.

[0003] Enzyme-linked immunosorbent assay (ELISA) is simple, rapid, and high-throughput, and is widely used in the detection of residual HCPs, such as the determination of the residual content of bovine serum albumin in the test sample in the 2015 edition of the Chinese Pharmacopoeia, and the determination of the residual amount of bacterial protein in the recombinant products produced by the Escherichia coli expression system in the 2020 edition of the Chinese Pharmacopoeia. However, ELISA requires specific antibody labeling and is only suitable for the detection of two or fewer protein contents, and cannot cope with the detection of the content of a mixture of multiple impurity proteins. HPLC can also be used to detect proteins, but due to different fermentation processes, there are many types of impurity proteins in high-molecular-weight glucose polymers, and some low-content impurity proteins cannot be detected at the same time, and the detection sensitivity is low.

[0004] The basic unit of protein is amino acid. The prior art CN202111067782.7 provides a method for determining the molar extinction coefficient of protein. The method uses a pre-column derivatization method and an ultra-high performance liquid chromatography to measure the amino acid content, calculate the molar extinction coefficient of the protein, and then calculate the concentration of the protein to be measured, and provides the following calculation method:

[0005]

[0006] However, this method is applicable to samples with only one protein, or a protein with high purity (>85%) in the sample, and the theoretical mass percentage (%) of the reference amino acid is generally obtained by experience, so this method lacks accuracy and is not applicable to the determination of residual protein in high molecular glucose polymers. In addition, there are fewer residual impurities in high molecular glucose polymers, and the amino acids that can be hydrolyzed are also less, usually not exceeding 10ppm, and the detection limit and quantitative limit of the existing methods cannot meet the above detection requirements. Therefore, it is urgent to develop a trace analysis method for amino acids of impurities. Summary of the invention

[0007] The present invention provides a method for amino acid trace analysis of impurity proteins in glucose polymers. The method can effectively determine the contents of 18 kinds of amino acids in acid hydrolyzed mixed impurity proteins. The method has high sensitivity and accuracy, a wide detection range and a low detection limit. The detection method is specific and reliable, and has universal applicability, and plays a positive role in the safety research of such raw materials or pharmaceutical excipients.

[0008] The invention provides a method for analyzing trace amounts of amino acids of impurity proteins in glucose polymers. The method comprises solid phase extraction, acid hydrolysis, pre-column derivatization and high performance liquid chromatography detection.

[0009] In some embodiments of the present invention, the solid phase extraction uses a silica-based C18 bonded phase column; the sample elution method of the solid phase extraction is water and acetonitrile elution; in some examples, the water and acetonitrile elution is eluted with a 50% v / v acetonitrile aqueous solution, or, water, 50% v / v acetonitrile aqueous solution, 70% v / v acetonitrile aqueous solution and acetonitrile are added in sequence for elution; in one example, the water and acetonitrile elution is eluted by adding water, 50% v / v acetonitrile aqueous solution, 70% v / v acetonitrile aqueous solution and acetonitrile in sequence; in one example, the silica-based C18 bonded phase column is a Waters Sep-PakVac6cc C18-500mg column; in some examples, the solid phase extraction also includes activating the column with an activator, optionally, the activator is methanol or ethanol, more preferably, methanol.

[0010] In some embodiments of the present invention, the solid phase extraction comprises the following steps:

[0011] S101. Take an appropriate amount of the test sample (i.e., glucose polymer), accurately weigh it, dissolve it in water and dilute it to prepare a sample solution;

[0012] S102. Take a silica-based C18 bonded phase column, i.e., a solid phase extraction (SPE) column, such as a Sep-pak C18 solid phase column, first add an appropriate amount of an activating agent, such as methanol, to activate the SPE column, then add an equal volume of water to wash the methanol, purify the sample solution by passing it through the SPE column several times, and discard the filtrate;

[0013] S103. First add water to rinse once, and discard the eluent; then add water, 50% v / v acetonitrile aqueous solution, 70% v / v acetonitrile aqueous solution, and 100% acetonitrile aqueous solution, i.e. acetonitrile, in sequence for elution, repeat 3 times, combine and collect the eluent, and concentrate the eluent with nitrogen.

[0014] In some embodiments of the present invention, the acid hydrolysis is carried out in a constant temperature drying oven at 115°C-125°C; in one example, the acid hydrolysis is carried out in a constant temperature drying oven at 120°C.

[0015] In some embodiments of the present invention, the acid hydrolysis is carried out using concentrated hydrochloric acid (concentration of 36wt% to 38wt%) of the same volume as the sample solution obtained by the solid phase extraction, and the solution after the acid hydrolysis is acid-base neutralized and concentrated; in some examples, the base used for the acid-base neutralization is sodium hydroxide solution, preferably, saturated sodium hydroxide solution. In some examples, the acid hydrolysis is carried out using concentrated hydrochloric acid of the same volume as the sample solution obtained by the solid phase extraction in a drying oven at 120°C for 20 to 24 hours, and the solution after the acid hydrolysis is acid-base neutralized and concentrated.

[0016] In some embodiments of the present invention, the pre-column derivatization is to add a boric acid buffer solution to the sample solution after acid hydrolysis, and then add a derivatization solution, seal it and react in a water bath in the dark; then, take it out and cool it to room temperature, dilute it to the scale with a phosphate buffer solution, shake it well, and centrifuge it to obtain the test solution; in some embodiments, the boric acid buffer solution is prepared from one part of 0.2 mol / L boric acid solution and 3 to 5 parts of 0.05 mol / L sodium tetraborate solution, and its pH is 9.0; in some embodiments, the pH of the phosphate buffer solution is 7.0.

[0017] In some embodiments of the present invention, the derivatization solution is a 2,4-dinitrofluorobenzene solution, preferably, a 2,4-dinitrofluorobenzene acetonitrile solution, more preferably, a 1 wt % 2,4-dinitrofluorobenzene acetonitrile solution.

[0018] In some embodiments of the present invention, the pre-column derivatization comprises the following steps:

[0019] S201. Completely transfer the solution to be derivatized to a 50 mL brown volumetric flask, accurately add 10 mL of pH 9.0 boric acid buffer solution, then add 10 mL of the derivatization solution, shake well, seal with a sealing film, place in a water bath, and react in the dark for a while;

[0020] S202. The solution treated in S201 was cooled to room temperature, diluted to scale with pH 7.0 phosphate buffer solution, shaken, and centrifuged to obtain a test solution;

[0021] Further, the boric acid buffer solution is prepared by 1 part of 0.2 mol / L boric acid solution and 3 to 5 parts of 0.05 mol / L sodium tetraborate solution;

[0022] Furthermore, the derivatization solution is a 1 wt % 2,4-dinitrofluorobenzene (DNFB) acetonitrile solution.

[0023] In some embodiments of the present invention, the detection conditions of the high performance liquid chromatography are: using a Shim-pack-scepter C18 chromatographic column; mobile phase A is 20mmol / L to 500mmol / L sodium acetate buffer, pH value is 6.0 to 7.5, mobile phase B is 40% v / v to 55% v / v acetonitrile aqueous solution; column temperature is 27°C; detection wavelength is 251nm to 370nm; mobile phase elution mode is:

[0024]

[0025] Further, the filler particle size of the chromatographic column is selected from 0.7 to 5 μm, the column length is selected from 50 to 250 mm, and the inner diameter is 4.6 mm;

[0026] Furthermore, the filler has a particle size of 3 μm.

[0027] Furthermore, the column length is 150 mm.

[0028] Furthermore, the mobile phase A is a 50 mmol / L sodium acetate buffer, and the buffer solution further comprises phosphoric acid and / or acetic acid, and N,N-dimethylformamide; wherein the phosphoric acid and / or acetic acid adjust the pH of the buffer solution.

[0029] Further, the pH of the mobile phase A is 6.3-7.0; preferably, the pH of the mobile phase A is 6.8;

[0030] Further, the concentration of N,N-dimethylformamide in mobile phase A is 0.01%;

[0031] Further, the mobile phase B is a 50% v / v acetonitrile aqueous solution;

[0032] Further, the flow rate is 0.7 mL / min;

[0033] Furthermore, the wavelength is selected from 360 nm.

[0034] The method for detecting the content of impurity proteins in a glucose polymer described in the present invention is applicable to the detection standard of the content of impurity proteins in a glucose polymer, wherein the glucose polymer is dextran, optionally, dextran 10, dextran 20, dextran 40, dextran 60, dextran 70, or dextran 75.

[0035] In an embodiment of the present invention, the amino acid analyzed or detected is the final result of processing by the present method.

[0036] In an embodiment of the present invention, the amino acids in the test sample, i.e., the glucose polymer, contain at least 7 of 18 kinds, and the 18 kinds of amino acids are aspartic acid, glutamic acid, serine, arginine, glycine, threonine, proline, alanine, valine, methionine, cystine, isoleucine, leucine, tryptophan, isophenylalanine, histidine, lysine and tyrosine; and the 7 kinds of amino acids can be threonine, glutamic acid, glycine, proline, alanine, methionine and tyrosine.

[0037] The wt% represents the weight percentage of the substance in the total substance.

[0038] The %, v / v and %(v / v) represent the volume percentage of the liquid in the total volume of the system.

[0039] The analytical method of the invention is accurate and reliable, has low quantitative limit concentration and detection limit concentration, high method sensitivity, and is suitable for amino acid analysis of trace impurity proteins in high-molecular glucose polymers.

[0040] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0042] Figure 1 is the chromatogram of blank derivatization solution;

[0043] Figure 2 This is the chromatogram of the derivatization solution of the reference substance. DETAILED DESCRIPTION

[0044] For ease of understanding of the present invention, the present invention will be described in detail below in conjunction with examples, but it is not intended to be limiting of the present invention. Unless otherwise specified, the professional terms used below are the meanings commonly understood by those of ordinary skill in the art. Unless otherwise specified, various raw materials, reagents, instruments and equipment of the present invention can be purchased from the market or prepared on site.

[0045] Experimental Example 1 Comparison of Solid Phase Extraction Columns

[0046] This method first screened different types of solid phase extraction columns. The specific experiments are as follows:

[0047] Reagents and controls:

[0048] Sodium Acetate, AR Grade

[0049] N,N-Dimethylformamide, GC grade

[0050] Concentrated hydrochloric acid, AR grade

[0051] Methanol, HPLC grade

[0052] Acetonitrile, HPLC grade

[0053] Boric acid, AR grade

[0054] Sodium Tetraborate, AR Grade

[0055] Phosphoric acid, AR grade

[0056] Sodium Hydroxide, AR Grade

[0057] 2,4-Dinitrofluorobenzene, AR grade

[0058] 18 amino acid reference substances, spectral grade

[0059] Preparation of test sample derivatization solution:

[0060] S1. Take 10 g of dextran 40 (batch number 105-20220201, Shanghai Huamao Pharmaceutical Co., Ltd.), accurately weigh, add water to dissolve and dilute to make a 0.4 mg / mL dextran solution.

[0061] S2. Take an SPE column (Agela PEP column based on (functionalized) polystyrene / divinylbenzene; Welchrom PS / DVB column based on highly cross-linked polystyrene / divinylbenzene copolymer; Waters Sep-Pak Vac 6cc C18-500mg, silica-based C18 bonded phase), add about 12 mL of methanol for activation, and then wash the methanol with an equal volume of water.

[0062] S3. Purify 25 mL of sample solution by SPE column in batches, and discard the filtrate. Then, add 5 mL of water for elution, discard the eluent, and then add 5 mL of water, 50% v / v acetonitrile (aqueous solution), 70% v / v acetonitrile (aqueous solution), and 100% acetonitrile for elution. Repeat 3 times, collect the eluate, and concentrate the eluate to about 5 mL.

[0063] S4. Add an equal volume of concentrated hydrochloric acid to the concentrated eluate of S3, seal the tube, and perform acid hydrolysis in a constant temperature drying oven at 120°C for 24 hours.

[0064] S5. Add an appropriate amount of saturated sodium hydroxide solution to the acid-hydrolyzed solution of S4 to neutralize to pH = 7, and concentrate to about 10 mL.

[0065] S6. Completely transfer the solution obtained in S5 to a 50 mL brown volumetric flask, accurately add 10 mL of pH 9.0 boric acid buffer solution, then accurately add 10 mL of 2,4-dinitrofluorobenzene derivatization solution (1 wt% 2,4-dinitrofluorobenzene in acetonitrile solution), shake well, seal with a sealing film, place in a 60°C water bath, and react in the dark for 60 minutes. Take out and cool to room temperature, then dilute to scale with pH 7.0 phosphate buffer solution, shake well, and the test sample derivatization solution is obtained.

[0066] Preparation of blank derivatization solution:

[0067] Accurately measure 5 mL of pH 9.0 boric acid buffer solution, place it in a 25 mL brown volumetric flask, add 5 mL of 2,4-dinitrofluorobenzene derivatization solution (1 wt% 2,4-dinitrofluorobenzene in acetonitrile solution), shake well, seal with sealing film, place in a 60°C water bath, and react in the dark for 60 minutes. After taking out, cool to room temperature, dilute to scale with pH 7.0 phosphate buffer solution, shake well, and obtain a blank derivatization solution.

[0068] Preparation of reference substance derivatization solution:

[0069] Reference substance stock solution: Take 15 mg of 18 kinds of amino acid reference substances, weigh accurately, place them in 18 50 mL volumetric flasks respectively, dissolve and dilute to the scale with pH 9.0 boric acid buffer, and shake well.

[0070] Reference solution: Accurately measure 1 mL of each amino acid reference stock solution, place in a 20 mL volumetric flask, dilute to the mark with pH 9.0 boric acid buffer, and shake well.

[0071] Accurately measure 5 mL of amino acid reference solution and pH 9.0 boric acid buffer solution, place in the same 25 mL brown volumetric flask, accurately add 10 mL of 2,4-dinitrofluorobenzene derivatization solution (1 wt% 2,4-dinitrofluorobenzene in acetonitrile solution), shake well, seal with sealing film, place in a 60°C water bath, and react in the dark for 60 minutes. After taking out, cool to room temperature, dilute to scale with pH 7.0 phosphate buffer solution, and shake well to obtain the reference derivatization solution.

[0072] HPLC detection:

[0073] Chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel (Shim-pack-scepter C18, 4.6 mm × 150 mm, 3 μm); mobile phase A: 50 mmol / L sodium acetate buffer (containing 0.01% N, N-dimethylformamide and adjusted to pH 6.8 with phosphoric acid); mobile phase B: 50% (v / v) acetonitrile aqueous solution; flow rate was 0.7 mL / min, detection wavelength was 360 nm, column temperature was 27°C, and injection volume was 20 μl. The gradient elution program is shown in Table 1.

[0074] Table 1. Gradient elution program

[0075] Time (min) Mobile phase A (%, v / v) Mobile phase B (%, v / v) 0 84 16 4 69 31 8.5 64 36 17 45 55 28 35 65 42 0 100 43 84 16 50 84 16

[0076] Qualitative and quantitative analysis :

[0077] The blank solution chromatogram is used for qualitative analysis of the sample. Figure 1 shown.

[0078] The chromatogram of the reference substance is used for qualitative and quantitative analysis of the sample. Figure 2 shown.

[0079] According to the detection chromatograms of blank solution, reference solution and test solution, the content of each amino acid in the test samples treated with different SPE was calculated by external standard method. The formula is:

[0080]

[0081] Where:

[0082] C x is the amino acid concentration of the test solution;

[0083] A x is the peak area of ​​amino acids in the test solution;

[0084] C r is the concentration of amino acid in the reference solution;

[0085] A r is the amino acid peak area in the reference solution.

[0086] Test results:

[0087] According to the chromatograms of the test and reference products, the effects of different SPE columns on the amino acid content of impurity proteins in dextran 40 (batch number 105-20220201) after acid hydrolysis were calculated. The results are shown in the following table:

[0088] Table 2 Effect of different SPE columns on the detected amino acid content

[0089]

[0090] According to the test results: different solid phase extraction columns have different effects on the enrichment and purification of impurities. According to the differences in the detected amino acid content and types, it can be found that for the same batch of test samples, the test samples treated with Waters Sep-pak Vac 6cc small column can detect 7 amino acids, and the content of each amino acid is also higher than that of the test samples treated with the other two extraction columns.

[0091] Experimental Example 2 Comparison of Solid Phase Extraction Column Activation Solvents

[0092] The Waters Sep-Pack Vac 6cc C18-500mg column of Experimental Example 1 was used as a solid phase extraction column to investigate the effects of different activated solvents on amino acid detection. The detection method is as follows: in the S2 step of the preparation of the test sample derivatization solution, purified water, acetonitrile, and ethanol were used to replace the methanol in Experimental Example 1, and the same batch (batch number 105-20220201) of dextran 40 produced by Shanghai Huamao Pharmaceutical Co., Ltd. was used as the sample. The rest of the detection method was the same as that of Experimental Example 1, and the results are shown in the following table:

[0093] Table 3 Effect of different SPE column activation conditions on the detected amino acid content

[0094]

[0095] According to the test results: different solid phase extraction column activation methods have a great influence on the amino acid detection after acid hydrolysis of impurity proteins. Among them, the activation effects of purified water, acetonitrile and ethanol are far inferior to those of methanol. The enrichment effect of the activated SPE column is poor, and there are interference peaks in the amino acid chromatogram, so the test results cannot be obtained or the test results are poor.

[0096] Experimental Example 3 Comparison of elution methods of solid phase extraction columns

[0097] Waters Sep-Pack Vac 6cc C18-500mg small column was used as a solid phase extraction column, and methanol was used as an activation solvent to investigate the effects of different elution methods on amino acid detection. The detection method is as follows: in the S3 step of the preparation of the test sample derivatization solution, purified water, acetonitrile, ethanol, and 50% acetonitrile (acetonitrile-water, 1:1 v / v) were used to replace the linear elution program in Experimental Example 1, and the same batch (batch number 105-20220201) of dextran 40 produced by Shanghai Huamao Pharmaceutical Co., Ltd. was used as the test sample. The rest of the detection method was the same as in Experimental Example 1. The amino acid detection results are shown in the following table:

[0098] Table 4 Effect of different SPE column elution methods on the detected amino acid content

[0099]

[0100] According to the test results: after elution with purified water, acetonitrile and ethanol, the types of amino acids in the test sample are obviously less than those in 50% acetonitrile and linear elution methods; while in 50% acetonitrile and linear elution methods, the types of amino acids and the content of amino acids in the latter are obviously higher than those in the former.

[0101] Experimental Example 4 Comparison of acid hydrolysis methods of impure proteins

[0102] Waters Sep-Pack Vac 6cc C18-500mg small column was used as solid phase extraction column, methanol was used as activation solvent, and linear elution mode was used to investigate the effect of different protein acid hydrolysis methods on amino acid detection. The detection method is: S4 step of preparation of the test sample derivatization solution, using 100℃ water bath; 120℃ oil bath instead of 120℃ constant temperature drying oven acid hydrolysis in Experimental Example 1, using the same batch (batch number 105-20220201) of dextran 40 produced by Shanghai Huamao Pharmaceutical Co., Ltd. as the test sample, the rest of the detection method is the same as Experimental Example 1, and the amino acid detection results are shown in the following table:

[0103] Table 5 Effects of different impurity protein acid hydrolysis methods on the detected amino acid content

[0104]

[0105] According to the test results: the types and contents of amino acids obtained by acid hydrolysis in a 120°C constant temperature drying oven are higher than those obtained by acid hydrolysis in a 100°C water bath and a 120°C oil bath.

[0106] Experimental Example 5 Comparison of HPLC Detection Conditions

[0107] The reference substance derivatization solution prepared in Experimental Example 1 was used to investigate the effects of the chromatographic column, column temperature, and elution method on the separation of different amino acids. The investigation method was a single factor investigation, and other HPLC detection methods remained unchanged, the same as in Example 1. The detection results were as follows:

[0108] Table 6 Effect of different chromatographic columns on amino acid separation

[0109]

[0110]

[0111] NA means approaching infinity, because tyrosine is the last sample to elute, and there is no next sample peak to form an effective separation with it, and the chromatograph integration result is displayed as NA.

[0112] Table 7 Effect of different column temperatures on amino acid separation

[0113]

[0114]

[0115] Table 8 Effect of different elution modes on amino acid separation

[0116]

[0117] Among them, the elution program of gradient elution 1 is as follows:

[0118]

[0119]

[0120] According to the test results of the derivatization solution of the reference substance, by selecting the Shim-pack Scepter-C18 chromatographic column, maintaining 27° C. and adopting the gradient elution described in Experimental Example 1, effective separation of different amino acids was achieved.

[0121] Example 1 Detection of trace amounts of amino acids in different batches of dextran 40

[0122] Two batches of dextran 40 produced by Shanghai Huamao Pharmaceutical Co., Ltd. were used as test samples. The sample processing method, chromatographic analysis conditions and detection method determined in the above Experimental Examples 1-5 were used. The test results are shown in the following table:

[0123] Table 9 Test results of amino acid content of impurity proteins in different batches of products from Shanghai Huamao Pharmaceutical Co., Ltd.

[0124]

[0125] Similarly, three batches of dextran 40 produced by Xi'an Wanlong Pharmaceutical Co., Ltd. were used as test samples, and the test results are shown in the following table:

[0126] Table 10 Test results of amino acid content of impurity proteins in different batches of products of Xi'an Wanlong Pharmaceutical Co., Ltd.

[0127]

[0128] Example 2 Detection of trace amounts of amino acids of impure proteins in different types of dextran

[0129] The amino acid content was tested using different types of dextran produced by Shandong Xiya Chemical Industry Co., Ltd., including dextran 20 and dextran 70. The sample processing method and the high performance liquid phase analysis method were as in Example 1. The test results are shown in the following table:

[0130] Table 11 Test results of amino acid content of impurity protein in different batches of products from Shandong Xiya Chemical Industry Co., Ltd.

[0131]

[0132]

[0133] 18 kinds of amino acid standard solutions were precisely added to the dextran samples of batches 102-20211001 and CC-20220716 described in Example 1, and batches 20230109 and 20221209 described in Example 2. The results are shown in the following table:

[0134] Table 12 Accuracy investigation results of the analytical method of the present invention (102-20211001 batch spiked results)

[0135]

[0136] From the results of spiked samples, we can see that this method can accurately detect 18 kinds of amino acids contained in the sample. Therefore, it can be determined that the types and contents of amino acids detected in the above table are accurate and reliable.

[0137] Table 13 Accuracy investigation results of the analytical method of the present invention (CC-20220716 batch spiked results)

[0138]

[0139] From the results of spiked samples, we can see that this method can accurately detect the 18 kinds of amino acids contained in the sample. Therefore, it can be determined that the types and contents of amino acids detected in the above table are accurate and reliable.

[0140] Table 14 Accuracy investigation results of the analytical method of the present invention (20230109 batch spiked results)

[0141]

[0142] Table 15 Accuracy investigation results of the analytical method of the present invention (20221209 batch spiked results)

[0143]

[0144]

[0145] From the results of spiked samples, we can see that this method can accurately detect 18 kinds of amino acids contained in the sample. Therefore, it can be determined that the types and contents of amino acids detected in the above table are accurate and reliable.

[0146] Linearity test of the method of the present invention

[0147] Prepare aspartic acid, glutamic acid, serine, arginine, glycine, threonine, proline, alanine, valine, methionine, cystine, isoleucine, leucine, tryptophan, phenylalanine, histidine, lysine, and tyrosine reference substance stock solutions in 10 mL volumetric flasks respectively: Accurately weigh a certain amount of various amino acid reference substances into a 10 mL volumetric flask, dissolve them with pH 9.0 boric acid buffer, and shake to volume.

[0148] Linearity 1 solution (5000% limit concentration, 50 μg / mL):

[0149] Component 1: Accurately measure 1.25 mL of the reference stock solution of aspartic acid, serine, glycine, tryptophan, lysine hydrochloride, and tyrosine into a 10 mL volumetric flask, dissolve it with pH 9.0 boric acid buffer, and shake it to volume. Take another 5 mL and place it in a 25 mL brown volumetric flask, then add 5 mL of pH 9.0 boric acid buffer solution and 5 mL of 2,4-dinitrofluorobenzene derivatization solution (1 wt% 2,4-dinitrofluorobenzene in acetonitrile), shake it well, seal it with a sealing film, put it in a 60°C water bath, and react in the dark for 60 minutes. Take it out, cool it to room temperature, and make up to volume with pH 7.0 phosphate buffer and shake it well.

[0150] Component 2: Accurately measure 1.25 mL of each reference stock solution of glutamic acid, threonine, alanine, valine, isoleucine, and phenylalanine into a 10 mL volumetric flask. Subsequent treatment is the same as that of linear 1-component 1 solution.

[0151] Component 3: Accurately measure 1.25 mL of arginine, proline, methionine, cystine, leucine, and histidine into a 10 mL volumetric flask. Subsequent treatment is the same as the linear 1-component 1 solution.

[0152] Linear 2 solution (4000% limit concentration, 40 μg / mL):

[0153] Accurately measure 2 mL of linear 1 solution, add 0.5 mL of pH 7.0 phosphate buffer solution, and mix well.

[0154] Linearity 3 solution (2500% limit concentration, 25 μg / mL):

[0155] Accurately measure 1 mL of linear 1 solution, add 1 mL of pH 7.0 phosphate buffer solution, and mix well.

[0156] Linear 4 solution (1000% limit concentration, 10 μg / mL):

[0157] Accurately measure 1 mL of linear 1 solution and place it in a 5 mL volumetric flask. Add pH 7.0 phosphate buffer solution to make up to volume and shake well.

[0158] Linear 5 solution (100% limit concentration, 1 μg / mL):

[0159] Accurately measure 1 mL of linear 4 solution and place it in a 10 mL volumetric flask. Add pH 7.0 phosphate buffer solution to make up to volume and shake well.

[0160] Linear 6 solution - (10% limit concentration, 0.1 μg / mL):

[0161] Accurately measure 1 mL of linear 5 solution and place it in a 10 mL volumetric flask. Add pH 7.0 phosphate buffer solution to make up to volume and shake well.

[0162] Linear 7 solution - (0.5% limit concentration, 0.005 μg / mL):

[0163] Accurately measure 1 mL of linear 6 solution and place it in a 20 mL volumetric flask. Add pH 7.0 phosphate buffer solution to make up to volume and shake well.

[0164] According to the chromatographic conditions of Example 1, each component solution of Linear 1 to Linear 7 was injected once, and the standard curve and correlation coefficient were obtained by the least squares linear regression of amino acid content and peak area.

[0165] Table 16. Standard curve and correlation coefficient

[0166]

[0167]

[0168] The above results show that the 18 amino acids detected show good linearity in the detection range of 0.05-50 μg / mL, indicating that the detection method of the present invention has high sensitivity, and the linear range of detection covers a range of 1000 times, and the concentration range covered is also relatively low. However, the linear range of the detection methods recorded in the prior art, such as prior art 1: CN103837620A (publication date: 2014.06.04), prior art 2: CN111398436A (publication date: 2020.07.10), prior art 3: Yang Qingshan et al., HPLC pre-column derivatization method for determining the content of 17 amino acids in cottonseed protein (2020) does not exceed 200 times (200 times can be calculated based on the known data disclosed in the prior art).

[0169] Investigation on the quantitative limit and detection limit of the method of the present invention

[0170] Blank solution: Accurately measure 5 mL of pH 9.0 boric acid buffer solution and place it in a 25 mL brown volumetric flask, then add 5 mL of 2,4-dinitrofluorobenzene derivatization solution (1 wt% 2,4-dinitrofluorobenzene in acetonitrile solution), shake well, seal with sealing film, place in a 60°C water bath, and react in the dark for 60 minutes. Take out, cool to room temperature, and dilute to volume with pH 7.0 phosphate buffer solution and shake well.

[0171] Aspartic acid reference stock solution: Accurately weigh 18.46 mg of aspartic acid into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0172] Glutamic acid reference stock solution: Accurately weigh 12.63 mg of glutamic acid into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0173] Serine reference stock solution: Accurately weigh 19.89 mg of serine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer, and shake to volume.

[0174] Arginine reference stock solution: Accurately weigh 19.64 mg of arginine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0175] Glycine reference stock solution: Accurately weigh 15.11 mg of glycine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0176] Threonine reference stock solution: Accurately weigh 17.25 mg of threonine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0177] Proline reference stock solution: Accurately weigh 15.52 mg of proline into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0178] Alanine reference stock solution: Accurately weigh 20.50 mg of alanine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0179] Valine reference stock solution: Accurately weigh 18.49 mg of valine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0180] Methionine reference stock solution: Accurately weigh 17.33 mg of methionine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0181] Cystine reference stock solution: Accurately weigh 15.97 mg of cystine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0182] Isoleucine reference stock solution: Accurately weigh 17.26 mg of isoleucine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer, and shake to volume.

[0183] Leucine reference stock solution: Accurately weigh 16.42 mg of leucine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer, and shake to volume.

[0184] Tryptophan reference stock solution: Accurately weigh 19.05 mg of tryptophan into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0185] Phenylalanine reference stock solution: Accurately weigh 15.85 mg of phenylalanine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0186] Histidine reference stock solution: Accurately weigh 18.90 mg of histidine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0187] Lysine hydrochloride reference stock solution: Accurately weigh 16.74 mg of lysine hydrochloride into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0188] Tyrosine reference stock solution: Accurately weigh 19.62 mg of tyrosine into a 50 mL volumetric flask, dissolve it in pH 9.0 boric acid buffer and shake to volume.

[0189] Component 1: Aspartic acid, serine, glycine, tryptophan, lysine hydrochloride, tyrosine

[0190] Component 2: Glutamic acid, threonine, alanine, valine, isoleucine, phenylalanine

[0191] Component 3: Arginine, Proline, Methionine, Cystine, Leucine, Histidine

[0192] Reference substance stock solution (component 1): Accurately measure 0.3 mL of the amino acid reference substance stock solution of component 1 into a 20 mL volumetric flask, dilute to volume with pH 9.0 boric acid buffer and shake well. Take another 5 mL and place it in a 25 mL brown volumetric flask, then add 5 mL of pH 9.0 boric acid buffer solution and 5 mL of 2,4-dinitrofluorobenzene derivatization solution (1 wt% 2,4-dinitrofluorobenzene in acetonitrile solution), shake well, seal with sealing film, place in a 60°C water bath, and react in the dark for 60 minutes. Take out, cool to room temperature, dilute to volume with pH 7.0 phosphate buffer and shake well.

[0193] Reference substance stock solution (component 2): Accurately measure 0.3 mL of the amino acid reference substance stock solution of component 2 into a 20 mL volumetric flask, dilute to volume with pH 9.0 boric acid buffer and shake well. The derivatization treatment is the same as that of the reference substance stock solution (component 1).

[0194] Reference substance stock solution (component 3): Accurately measure 0.3 mL of the amino acid reference substance stock solution of component 3 into a 20 mL volumetric flask, dilute to volume with pH 9.0 boric acid buffer and shake well. The derivatization treatment is the same as that of the reference substance stock solution (component 1).

[0195] Quantitative limit concentration solution (1% limit concentration, i.e. 1 μg / mL): Accurately measure 1 mL of each reference substance stock solution (component 1, component 2, component 3) and place them in 100 mL volumetric flasks, dilute to volume with pH 7.0 phosphate buffer solution and shake well.

[0196] Detection limit concentration solution (0.3% limit concentration): Accurately measure 3 mL of the quantitation limit solution and place it in a 10 mL volumetric flask, dilute to volume with pH 7.0 phosphate buffer solution and shake well.

[0197] The liquid chromatography conditions of Example 1 were adopted, and the quantitative limit concentration solution was sampled for 6 consecutive injections, and the detection limit concentration solution was sampled for 1 injection. The test results are shown in Table 7: The quantitative limit and detection limit concentrations of the 18 amino acids tested (aspartic acid, serine, glycine, tryptophan, lysine hydrochloride, tyrosine, glutamic acid, threonine, alanine, valine, isoleucine, phenylalanine, arginine, proline, methionine, cystine, leucine, histidine) are shown in the following table, and the analysis method of the present invention is listed with the prior art 1: CN103837620A (publication date: 2014.06.04), prior art 2: CN111398436A (publication date: 2020.07.10), prior art 3: Yang Qingshan et al., HPLC pre-column derivatization method for determining the content of 17 amino acids in cottonseed protein (2020):

[0198] Table 17 Detection limit of the analytical method of the present invention and its comparison with the prior art

[0199]

[0200] Table 18 Quantitative limit of the analytical method of the present invention and comparison results with the prior art

[0201]

[0202] Comprehensive test results show that the detection method of the present invention has high sensitivity and accuracy.

[0203] In summary, the detection method provided by the present invention can achieve accurate quantification of 18 kinds of amino acids that may be contained in the protein of dextran produced by different preparation processes, and can meet the quality control and finished product inspection in the dextran production process.

Claims

1. A method for analyzing trace amounts of amino acids in impurity proteins in glucose polymers, the method comprising solid phase extraction, acid hydrolysis, pre-column derivatization and high performance liquid chromatography detection, wherein: The solid phase extraction uses a silica-based C18 bonded phase column; the sample elution method of the solid phase extraction is water and acetonitrile elution; The acid hydrolysis method is acid hydrolysis in a constant temperature drying oven at 115°C-125°C; The HPLC detection conditions are as follows: Shim-pack-scepter C18 chromatographic column is used; mobile phase A is 20mmol / L to 500mmol / L sodium acetate buffer, pH value is 6.0 to 7.5, mobile phase B is 40% v / v to 55% v / v acetonitrile aqueous solution; column temperature is 27°C; detection wavelength is 251nm to 370nm; mobile phase elution mode is:

2. The analysis method according to claim 1, wherein The silica-based C18 bonded phase column is a Waters Sep-Pak Vac 6cc C18-500mg column.

3. The analysis method according to claim 1, wherein The solid phase extraction further comprises activating the small column with an activator, optionally, the activator is methanol or ethanol, more preferably, methanol; and / or The water and acetonitrile elution is performed by using a 50% v / v acetonitrile aqueous solution, or by sequentially adding water, a 50% v / v acetonitrile aqueous solution, a 70% v / v acetonitrile aqueous solution and acetonitrile for elution; preferably, water, a 50% v / v acetonitrile aqueous solution, a 70% v / v acetonitrile aqueous solution and acetonitrile are sequentially added for elution.

4. The analysis method according to claim 1, wherein The acid hydrolysis is carried out by treating the sample solution in a drying oven at 120° C. for 20 to 24 hours using concentrated hydrochloric acid of an equal volume to the sample solution obtained by the solid phase extraction, and the solution after the acid hydrolysis is neutralized with acid and alkali and concentrated.

5. The analysis method according to claim 1, wherein The pre-column derivatization is to add a boric acid buffer solution to the sample solution after acid hydrolysis, and then add a derivatization solution, seal it and react in a dark water bath; then, take it out and cool it to room temperature, dilute it to the scale with a phosphate buffer solution, shake it well, and centrifuge it to obtain the test solution.

6. The analysis method according to claim 5, wherein: The boric acid buffer solution is prepared from one part of 0.2 mol / L boric acid solution and 3 to 5 parts of 0.05 mol / L sodium tetraborate solution, and has a pH of 9.0; and / or The pH of the phosphate buffer solution is 7.

0.

7. The analysis method according to claim 1, wherein The derivatization solution is a 2,4-dinitrofluorobenzene solution, preferably, an acetonitrile solution of 2,4-dinitrofluorobenzene, and more preferably, a 1 wt % 2,4-dinitrofluorobenzene acetonitrile solution.

8. The analysis method according to claim 1, wherein The mobile phase A is 50 mmol / L sodium acetate buffer with a pH value of 6.8; the mobile phase B is 50% v / v acetonitrile aqueous solution; and / or The mobile phase flow rate of the high performance liquid chromatography detection is 0.4-0.8 mL / min, preferably 0.7 mL / min; and / or The detection wavelength is 360nm; and / or The injection volume was 20 μL.

9. The analysis method according to claim 1, wherein: The filler particle size of the chromatographic column is 0.7-5 μm, preferably 3 μm; the column length is 50-250 mm, preferably 150 mm; and the inner diameter is 4.6 mm.

10. The analysis method according to any one of claims 1 to 9, wherein: The glucose polymer is dextran, optionally, dextran 10, dextran 20, dextran 40, dextran 60, dextran 70, or dextran 75.

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